IDENTIFICATION OF A MEMBRANE-PROTEIN INVOLVED IN ACTIVATION OF THE KINB PATHWAY TO SPORULATION IN BACILLUS-SUBTILIS

The initiation of sporulation in Bacillus subtilis is dependent on the phosphorylation of the SpoOA transcription factor mediated by the pho sphorelay and by two major kinases, KinA and KinB. Temporal expression of these kinases was analyzed, and an assessment of their respective contributions to the production of SpoOA similar to P was undertaken. The results show that KinB is expressed and activated prior to KinA; i .e., the two kinases are solicited sequentially in the sporulation pro cess and are thought to be activated by different signaling pathways. A strategy was developed to isolate mutations specifically affecting t he KinB pathway, using the newly improved mini-Tn10 delivery vector pI C333. Several mutants were obtained, one of which carried a transposon in a gene coding for a small integral membrane protein, named KbaA. I nactivation of the kbaA gene appeared to affect KinB activity but not transcription of kinB. A Spo(+) suppressor (kinB45) of the kbaA null m utation was isolated in the promoter region of kinB. An eightfold incr ease of kinB expression levels over wild-type levels was observed in t he kinB45 mutant. Thus, overexpression of the kinB-kapB operon was suf ficient to overcome the sporulation defect caused by inactivation of k baA in a KinA(-) strain. Transcription of kinB was found to be repress ed by SinR, while the kinB45 mutant was no longer sensitive to SinR re gulation. Implications of these observations on the transcriptional re gulation of kinB and the role of KbaA in KinB activation are discussed .